The rapid emergence of multi-drug resistant pathogenic microorganisms represents a major threat to public health, placing an ever-increasing demand for the discovery of new antibacterial agents. Macrolide antibiotics, such as the 14-membered macrolide erythromycin and second-generation analogs clarithromycin and azithromycin, are among the first line therapies clinically employed to treat respiratory tract infections. However, as a consequence of the clinical overuse of these agents, macrolide resistance mechanisms have rapidly emerged. In contrast, 16-membered macrolides have demonstrated the capability of overcoming resistance mechanisms that affect 14- and 15-membered macrolides. Indeed, a few select 16- membered macrolides have been successfully employed in the clinical treatment of bacterial infections outside of the United States. Despite their demonstrated potential, however, 16-membered macrolides still remain underexplored in the development of new human antibacterial agents. The tylosin biosynthetic pathway produces a series of 16-membered macrolides, of which the veterinary therapeutic tylosin is best known. In this SBIR proposal, Alluvium Biosciences proposes to genetically engineer the tylosin biosynthetic pathway to enable the production of novel 16-membered macrolide compounds for application in new antibiotic drug discovery. In this effort, a genetically engineered Streptomyces fradiae strain will be generated via genomic integration of the heterologous cytochrome P450 monooxygenase, mycG. This cytochrome P450 is native to the mycinamicin biosynthetic pathway that is responsible for the production of the mycinamicin family of 16- membered macrolides in Micromonospora griseorubida. It has previously been established that during the biosynthesis of the mycinamicins, MycG activity installs a regio- and stereospecific hydroxyl and/or epoxide functionality onto the mycinamicin core scaffold, resulting in both mono and di-oxidized bioactive compounds. It is established that this oxidative functionality is critical for mycinamicin bioactivity. Based on preliminary work, Alluvium expects that the engineered S. fradiae strain will be capable of producing hybrid tylosin-based analogs featuring an oxidation pattern similar to that observed in the mycinamicin family of macrolides. As it is known that the regio- and stereospecific oxidative functionalities can influence the bioactivity of macrolide compounds, Alluvium hypothesizes that the novel 16-membered macrolides will display potency against macrolide resistant bacterial pathogens. Accordingly, in vitro evaluation of antibacterial activities against a series of bacterial strains, including those that display macrolide resistance will be performed within this initial Phase I study. If the hypothesis is supported, compounds demonstrating promising activity will proceed to Phase II R&D wherein Alluvium will pursue medicinal chemistry efforts in order to optimize pharmacological properties and establish a lead macrolide antibiotic candidate.